The present invention relates to a process for detecting a water-soluble oxidized lipid. The process is capable of readily confirming that a specimen contains a water-soluble oxidized lipid. The present invention also concerns a process for forming a water-soluble lipid.
Oxidized lipids are believed to cause adverse effects in vivo, and have been pointed out to be responsible for arteriosclerosis, cancer, inflammation, aging, and other conditions. It is also believed that low density lipoproteins which account for a majority of lipids in blood are related to the above-mentioned disorders.
On the other hand, oxidized lipids which are controlled and produced by enzymes in vivo, such as prostaglandin and leukotriene, are likely to become substances having a strong influence on a living body.
Various methods for detecting a stable oxidized lipid or a final metabolite thereof have been developed; however, none of these methods permits satisfactory evaluation of the lipid oxidation process, this is partially due to difficulty in non-destructively isolating and differentiating specific oxidized lipids from other substances present in a sample that interfere With assays. These methods include, for example, the following: an iodine oxidation method which involves ionizing iodine by oxidation, followed by emission spectroscopic analysis on the ionized iodine; TBARS (thiobarbituric acid reactive substance) measuring method which involves producing a pigment by reaction with malondialdehyde as a peroxide lipid metabolite, followed by determination of the pigment by means of an absorption spectrophotometer or a fluorophtometer; conjugated diene determination method which involves determining a conjugated diene based on the fact that a conjugated diene is formed by peroxidation of an unsaturated fatty acid; high performance liquid chromatography (HPLC) method which involves separating and determining a peroxide lipid sample by means of a high performance liquid chromatograph; and gas chromatography method which involves esterifying a peroxide lipid sample, followed by separation and determination by means of gas chromatography.
However, the iodine oxidation method and the conjugated diene determination method which determine an oxidized lipid have a drawback in that they are not suitable for analysis of a sample containing an impurity. Furthermore, the iodine oxidation method, the TBARS method, and the conjugated diene determination method are non-specific determination methods, i.e., these methods do not directly determine an oxidized lipid per se, but indirectly determine an oxidized lipid by determining a by-product which is formed through formation or decomposition of an oxidized lipid.
On the other hand, the HPLC method and the gas chromatography method are specific methods which directly determine an oxidized lipid per se. In the HPLC method, however, it takes a long period of time to complete determination, so that a sample can undergo denaturation. In addition, an oxidized lipid per se is likely to undergo alteration by a solvent such as ethyl ether or ethanol. Furthermore, in the gas chromatography method, a sample is likely to undergo alteration through esterification treatment or the like.
Various methods for producing an oxidized lipid have heretofore been reported. Among these methods, there may be mentioned, for example, the following: a method which involves adding a transition metal such as copper cation (Cu.sup.+) or iron cation (Fe.sup.2+) to a lipid sample, and allowing the mixture to stand at a temperature of about 37.degree. C. for a period of 20 to 30 hours; a method which involves irradiating a sample, that is preliminarily added with a photosensitive substance, with light; and a method which comprises oxidation by an enzyme such as lipoxygenase or cyclooxygenase.
As described above, there has never been disclosed a method for precisely detecting and analyzing an oxidized lipid having physiological influence or an unstable oxidized lipid which adversely affects a body. In addition, there has not yet been developed a method for forming a water-soluble oxidized lipid having a hydroperoxide group which has specific influence on a living body. Furthermore, a method for precisely detecting a water-soluble oxidized lipid having a hydroperoxide group has not been developed. Accordingly, it has been impossible to evaluate formation of a specific water-soluble oxidized lipid. Water-soluble oxidized lipids having a hydroperoxide group have potentiality to be used as pharmaceuticals or intermediates thereof. For example, the water-soluble oxidized lipid can be used as an anticarcinogen when it comes into contact (e.g., via injection) with cancer cells since such oxidized lipids damage cells when provided with free radicals. The oxidized lipids can also be applied as a vasodilator for treatment of arterial infractions such as encephalic and myocardial infractions.
An object of the present invention was to solve the above-mentioned problems.